It has been suggested that starch (or dextrin)-degrading yeasts could be usefully exploited in fermentation processes, such as the production of ethanol or beer, which utilise starch-containing raw materials (Tubb, 1983, 1984: full reference to be found at the end of this specification), and in the production of amylolytic enzymes such as amyloglucosidases (Eveleigh, 1981; Fogarty, 1983). Dex.sup.+ strains of Saccharomyces cerevisiae (formerly called S. diastaticus; van der Walt, 1970; Yarrow, 1984) have at least one DEX or STA gene (Erratt and Stewart, 1978; Tamaki, 1978) and are able to ferment soluble starch or dextrins as a consequence of producing an extracellular amylo-.alpha.-1,4-glucosidase (AMG) during vegetative growth (Hopkins, 1955; Searle and Tubb, 1981). However, common brewing strains of Saccharomyces cerevisiae do not have the ability to produce extracellular AMG during vegetative growth. Dex.sup.+ strains of Saccharomyces cerevisiae have been hybridised with brewing strains, and progeny have been derived which have the ability to produce AMG during vegetative growth. However these progeny strains produce unacceptable low carbohydrate beers (Tubb et al., 1981), unless steps are taken to eliminate a gene (POFl) responsible for a `herbal phenolic` off-flavor (Goodey and Tubb, 1982).
Recombinant DNA techniques offer a more specific approach to conferring amylolytic character on strains of yeast already possessing many other desirable commercial characteristics. Recently, .alpha.-amylase genes from mice (Thomsen, 1983) and wheat (Rothstein et al., 1984) have been expressed in S. cerevisiae, and a gene for AMG production has been cloned from a STA1 strain of yeast (Yamashita and Fukui, 1983).
A 3.6kb DNA fragment has been cloned from a Saccharomyces diastaticus genome (strain BRG536): DEX1) and shown to confer production of extracellular amylo-.alpha.-1,4-glucosidase (AMG), and thereby, the ability to hydrolyse starch and dextrins, on Dex.sup.- strains of S. cerevisiae (Tubb, R. S., Brewers Guardian, Sept. 1984, 34-37). (A preliminary report of this work was given at the ALKO Symposium on gene expression in yeast at Helsinki in June, 1983).
The use of a eukaryotic signal sequence to promote product transport from a eukaryotic host cell harbouring a recombinant vector is known. Published European patent application EP-A1-0127304 describes fusion polypeptides comprising a signal (or "pre") sequence and a desired polypeptide produced by expression of a gene in a host cell. The fusion polypeptides are transported through the host cell membrane and cleaved to produce extracellular, mature, polypeptides. The yeast invertase signal sequence is specifically mentioned and its use in the preparation of host cells capable of producing extracellular interferon is described. Published European patent application EP-A1-0116201 describes an essentially similar use of eukaryotic signal sequences and exemplifies the use of the yeast .alpha.-factor signal sequence in the production of extracellular human epidermal growth factor (hEGF). The existence and use of the signal sequence of a yeast amylolytic enzyme to promote product secretion of heterologous polypeptides from yeast has been the subject of speculation (Tubb, R. S., Brewers Guardian, Sept. 1984, 34-37).
The cloned 3.6kb DNA fragment referred to above has now been sequenced and the coding sequence for AMG has been identified and elucidated. In addition, it has been unequivocally shown that the AMG gene includes a leader sequence coding for a signal peptide capable of promoting product secretion.
According to the present invention, there is provided a precursor polypeptide having the amino acid sequence:
met-gln-arg-pro-phe-leu-leu-ala-tyr-leu-val-leu-ser-leu-leu-phe-asn-ser-ala -leu-gly-X PA1 5'-ATGCAAGACCATTTCTACTCGCTTATTTGGTCCTTTCGCTTCTATTTAACTCAGCTTTGGGT(X.sup.1)- 3'
wherein X is a polypeptide.
The precursor polypeptide, when produced in a eukaryotic host cell by the expression of a gene coding for the precursor polypeptide, is exported from the host cell and processed to produce the mature polypeptide, X.
The polypeptide X may be any polypeptide such as an enzyme, a hormone, a lymphokine or the like. The mature polypeptide may itself be a precursor such as a pro form. Particular examples described herein are the yeast enzyme AMG, the mammalian enzyme, gastric lipase and the mammalian lymphokine, interferon-60 2. The amino acid sequences for the AMG and human gastric lipase are shown in FIGS. 4 and 5 respectively.
In a further aspect of the invention there is provided a DNA sequence coding for a precursor polypeptide according to the invention. The DNA sequence may be used to construct vectors for use in transforming eukaryotic host cells. The DNA sequence may have the following nucleotide sequence:
wherein X.sup.1 is the coding sequence of the polypeptide X.
In a further aspect of the invention, there is provided a eukaryotic expression vector including a DNA sequence of the invention positioned relative to a promoter capable of directing expression of the DNA sequence when the vector is transformed into a eukaryotic host cell, provided that when the polypeptide X is amylo-.alpha.-1,4-glucosidase, the promoter is not an amylo-.alpha.-1,4-glucosidase gene promoter.
The promoter may be any functionally active eukaryotic promoter but is preferably a yeast promoter, such as a promoter derived from the phosphoglycerate kinase (PGK) gene. The vector is adapted for expression in a eukaryotic host cell by the provision of selectable markers and control regions as, appropriate.
In a further aspect of the invention there is provided a eukaryotic host organism transformed with a vector of the invention. The host organism may be any eukaryotic organism including mammalian cells in tissue culture, but is preferably a yeast. Especially preferred are strains of Saccharomyces cerevisiae. Where the polypeptide X is an amlolytic enzyme, the yeast is preferably a brewing strain.
In a further aspect of the invention there is provided a fermentation process for producing ethanol comprising the step of culturing a yeast, in the presence of starch or dextrin, transformed with a vector of the invention including a gene coding for an amylolytic enzyme.
Such a fermentation will allow for the super-attenuation of wort liquor and consequently the production of a low-carbohydrate beer.
In a further aspect of the invention there is provided a process for the production of a polypeptide comprising growing, in a culture medium, a eukaryotic host organism transformed with a vector of the invention and isolating the polypeptide from the culture medium.